1.5 Enzymes

Question 1

Hwo does the binding of enzymes to transition states compare to binding of substrate or product?

Answer 1

• Enzymes bind transition states best
• Enzyme active sites are complimentary to the transition state of the reaction so they bind better than the substrates
• Additional interactions also lower activation barrier

Question 2

Answer 2

Question 3

How do enzymes lower the activation energy?

Answer 3

• Enzyme catalysed reactions are characterised by the formation of acomplex between enzyme and substrate
• Stabilisation of the transition state through tight binding of the enzyme

Question 4

What are the three catalytic mechanisms enzymes may take?

Answer 4

• Acid base catalysis - give and take protons
• Covalent catalysis - change reaction paths
• metal ion catalysis - use redox cofactors, pKa shifters

Question 5

Why are enzymes so important in metabolism?

Answer 5

Living organisms must be able to catalyse the conversion of carbon fuel sources into cellular energy (ATP) in an approopriate time scale

Question 6

What causes phenylketonuria?

Answer 6

A deficiency in the enzyme phenylalanine hydroxylase.

Question 7

What are cofactors and coenzymes?

Answer 7

• additional chemical components that enzymes require for activity
• Either small inorganic molecules called cofactors eg Mg²⁺, K
• More complex molecules called coenzymes that transiently carry funcitonal groups during catalysis of a reaction

Question 8

What are some of the most common types of enzymes?

Answer 8

Question 9

What are kinases?

Answer 9

They catalyse the phosphoryl transfer from one molecule (usually ATP) to another for example hexokinase

Question 10

What is the phosphorylase enzyme?

Answer 10

Catalyses the covalent addition of inorganic phosphate (Pi) to a moelcules

Eg glycogen phosphorylase

Question 11

What is the phosphatase enzyme?

Answer 11

It catalyses the cleavage of a phosphate to yield the dephosphorylated product and Pi

Example glucose-6-phosphatase

Question 12

What is the dehydrogenase enzyme?

Answer 12

It catalyses a redox reaction commonly using NADH/NAD+, NADPH/NADP+ or FADH₂/FAD as cofactors

Example glyceraldehyde-3-phosphate dehydrogenase

Question 13

What does the mutase enzyme do?

Answer 13

Catalyses the shift of a phosphoryl group from one atom to another within the same molecule

Example phosphoglycerate mutase

Question 14

What does the isomerase enzymes do?

Answer 14

Catalyses the conversion of one isomer to another, triose phosphate isomerase

Question 15

What does the hydratase enzyme type do?

Answer 15

Catalyses the addition/removal of water e.g enolase

Question 16

What does synthase enzyme do?

Answer 16

Catalyses the synthesis of a product.

Example citrate synthase

Question 17

Answer 17

Question 18

Answer 18

Question 19

What is the binding free energy?

Answer 19

The difference between the activation energies of the uncatalysed and catalysed reactions caused by the enzyme binding the transition state

Question 20

WHy does the rate of reaction decrease over time for enzyme substrate reaction?

Answer 20

• S is depleted by conversion to products
• The reaciton is reversible so as [P] increases the rate of the reverse reaction increases
• The enzyme may be unstable under the reaction conditions

Question 21

What is the relationship between the initial rate of reaction and the concentration of enzyme?

Answer 21

Question 22

How do you work out what the Km for an enzyme is?

Answer 22

1. Determine V_o at varying [S]
2. Plot V_o as a function of [S] and see the concentration of substrate at which V_o is half of Vmax

Question 23

In michaelis menten theory what is the form of the enzyme substrate reaction?

Answer 23

Question 24

What are the four assumptions used in michaelis menten theory?

Answer 24

Question 25

What is the michaelis menten equation?

Answer 25

Question 26

What is the rate determining step in michaelis menten kinetics?

Answer 26

The conversion of ES to E and P

Question 27

What is the steady state assumption for michaelis menten kinetics?

Answer 27

V_o represents the steady state where [ES] remains constant. Right when the reaction starts the rate of formation of ES = the rate of ES breakdown

Question 28

How does steady state assumption impact equilibria?

Answer 28

Question 29

What equation is used for the double reciprocal plot?

Answer 29

Question 30

What are the axis of each double reciprocal plot?

Answer 30

x axis is 1/[S] the y axis is 1/V_o

Question 31

What is on the intercepts of the double reciprocal plots?

Answer 31

X intercepts represents the -1/Km Y intercept is 1/V_max

Question 32

What is the slope of the double reciprocal plot?

Answer 32

Km/Vmax

Question 33

What is the relationship between Kd and Km?

Answer 33

Km has the Steady State assumption and takes account of the catalytic step

Question 34

What is the turnover number and where does it come from?

Answer 34

* In michaelis menten kinetics K₂ is the rate constant for the rate limiting step * Called K_cat or the turnover number * Means the number of molecules of substrate converted to product (S to P) per unit time per enzyme molecule saturated with substrate (when [ES] = [E_t]

Question 35

What is the specifity constant?

Answer 35

* It is K_cat/Km * Defined as the rate constant for the conversion of E+S to E+P * It reflects both substrate affinity and catalytic efficiency, high value indicates more efficient use of the substrate

Question 36

What are irreversible inhibitors defined as?

Answer 36

They bind covalently to the active site, destroy a functional group essential for enzyme activity, or form a stable non covalent complex with the enzyme (suicide inhibitors)

Question 37

What are reversible inhibitors defined as?

Answer 37

They bind reversibly to the enzymes and inhibit the enzyme either by competitive, uncompetitive or mixed modes of inhibition

Question 38

What changes in the michaelis menten equation are seen due to competitive inhibition?

Answer 38

There is an alpha constant added in front of Km

Question 39

How does the double reciprocal plot change for a competitive inhibition?

Answer 39

As alpha increase the gradient increases so x intercept gets closer to the right

Question 40

How does the michaelis menten equation change with uncompetitive inhibition?

Answer 40

And alpha' constant gets added in front of [S]

Question 41

How does the shape of the double reciprocal plot change with uncompetitive inhibition?

Answer 41

The line gets translated up the y axis with increasing alpha prime

Question 42

How does the michaelis menten equation change with the mixed inhibition?

Answer 42

alpha gets added to K_m and alpha' to [S]

Question 43

How does mixed inhibition change the shape of the double reciprocal plot?

Answer 43

Question 44

What do allosteric enzymes do?

Answer 44

* Regulate metabolic pathways by changing activity in response to chagnes in the concentration of molecules around them * Allosteric enzymes are regulated by compounds called allosteric modulators or allosteric effectors

Question 45

What are the types of allosteric modulators and how do they bind?

Answer 45

* Positive modulators’ activate and ‘negative modulators’ inhibit allosteric enzymes * Modulators bind reversibly and non-covalently to the enzyme – they can ‘come and go’

Question 46

How does a positive allosteric regulator work?

Answer 46

Question 47

How do allosteric regulators change the shape of V_o and [S] graph?

Answer 47

* As S (e.g.forATCase, S is Asp or carbamyl phosphate) binds, there is a transition from the T state to the R state to give a sigmoidal V0 versus S plot * A relatively small increase in S on the steep part of the curve causes a relatively large increase in V0

Question 48

What is the role of ATCase?

Answer 48

It catalyses the first step in the E coli pathway to produce the nucleotides UTP and finally CTP

Question 49

How is ATCase seen to be an allosteric enzyme?

Answer 49

* CTP inhibits ATCase at high levels so its a negative modulator * HIgh ATP levels in the bacteria indicate the growth of the cell and the need for more CTP, so ATP is a positive modulator

Question 50

What is the structure of ATCase?

Answer 50

It has complex quaternary structure of 12 subunits * 6 catalytic subunits, arranged as 2 x trimeric complexes. The catalytic subunits function cooperatively * 6 regulatory subunits, arranged as 3x dimeric complexes

Question 51

How does the shape of the V_o vs [S] plot change with modulators?

Answer 51

* As ATP increases, the V₀ vs [S] plot becomes more hyperbola-like * As CTP increases, the V0 vs [S] plot shifts to the right – the substrate concentration at 1⁄2 V₀ (K_0.5) increases